Bacillus thuringiensis subsp. israelensis (Bti) has been used in the field for over twenty years without resistance development in any target insect. In contrast, mosquito resistance to B. sphaericus has been observed in the field in many countries. This remarkable difference in the propensity to develop resistance to two mosquitocidal Bacillus strains is likely due to the presence of multiple toxins in Bti. However, mosquitoes are able to rapidly develop resistance to individual toxins from this strain. A major reason for the apparent inability of mosquitoes to develop resistance to Bti is the presence of cytolytic (Cyt) toxins in this and other mosquitocidal strains. During the last grant period we showed that key to the lack of mosquito resistance to Bti was how two toxins in this strain, namely Cry11A and Cyt1A toxins interact. We showed Cyt1A acts as a surrogate receptor for Cry11Aa. Importantly Cyt1Aa binds through Cry11A loop domains that are also involved in binding endogenous Aedes aegypti receptor proteins. Our research showed Aedes receptor proteins include cadherin, alkaline phosphatases (ALP), and aminopeptidases (APNs). All three classes bind mosquitocidal toxins with relatively high affinity, unlike in lepidopterans where only cadherin binds with high affinity. This implies both APN and ALP of mosquitoes could act as primary rather than as secondary receptors as in moths, suggesting a possible difference in the mode of action of mosquitocidal and lepidopteran Cry toxins. We believe however, there is conservation in the mode of action of Bt Cry toxins. Therefore we hypothesize a similar mode of action of action occurs in mosquitoes as in lepidopterans. In this proposal we plan to test this hypothesis. Consequently, we hypothesize that: (i) cadherin is a key protein which mediates initial binding to mosquitocidal Cry toxins and is essential for larval toxicity; ii) ALPs (and APNs) act as secondary receptors that allow toxin targeting to the cell membrane. Also in the previous proposal we showed Cyt1A plays a critical role in acting as a surrogate receptor for Cry11Aa. We therefore hypothesize that (iii) Cyt1A also is a receptor for other Cry toxins in Bti, and synergizes the toxicity of mosquitocidal Cry toxins. We will test this hypothesis and also elucidate the mechanism by which Cyt toxins insert into the membrane to act as surrogate receptors. This project is also a proposal to continue a successful collaboration between three different investigators to best use the expertise of each laboratory.